Proteases, which are enzymes that catalyze the hydrolysis of peptide bonds, play essential roles in numerous differnt biological processes and increasingly serve as important therapeutic targets. One powerful strategy for the development of protease inhibitors has been to design molecules that incorporate into the peptide substrate an isostere, or stable mimetic,of the transition state of the protease catalyzed reaction. Unfortunately, peptide-based drugs have had relatively limited utility as theapeutic agents due to poor adsorption and oral availability, rapid serum, clearing times, and/or rapid liver clearance and bilary excrtion. Relatively small inhibitors (less than 800 molecular weight) that display nonpeptide functionality about an appropriate isotere have therefore been the focus of efforts to improve pharmacokinetic properties, but many different compounds generally must be prepared and evaluated in an iterative process before small, nonpeptide inhibitors are identified that have high affinity. We propose to develop general methods for the rapid identification of low molecular weight, nonpeptide protease inhibitors using a combination of mechanism-based design, combinatorial synthesis and evaulation, and where structural data is available, structure-based design. We have first focused on the aspartic acid protease clss, for which human, parasite, fungi, and retroviral proteases have all served as important theapeutic targets. In preliminary studies we have demonstrated the power of the approach by identifying a number of low molecular weight, nonpeptide inhibitors of cathepsin D (Ki less than 15 nM), an aspartic acid protease that has been implicated in a number of disease states but for which nonpeptide inhibitors have not yet been reported [Science, submitted]. We will further demonstrate the approach by identifying potent and specific inhibitors of the malarial protease plasmepsin II, which is required by the parasite for degradation of host hemoglobin, and therefore may be an important therapeutic target for the treatment of malaria.